A Genetic Evaluation of the Leafnose Snake, Phyllorhynchus arenicolus

1980 JOURNAL OF HERPETOLOGY 14(3):26%268 A Genetic Evaluation of the Leafnose Snake, Phyllorhynchus arenicolus Robert W. Murphy and John R. Ottley Department of Biology, UCLA, Los Angeles, CA 90024 and Department of Herpetology, Monte L. Bean Life Science Museum, 290 MLBM, Brigham Young University, Provo, Utah 84602 ABSTRACT-A population of leafnose snakes from lsla Monserrate, Gulf of California, Mexico was recently described as Phyllorhynchus arenicolus Savage and Cliff. The nomenclatorial status of this population has since been questioned but without supportive evidence. This population was electrophoretically compared with populations of P. decurtatus on the peninsula of Baja California and with a population on a landbridge island, lsla San Marcos. The genetic comparisons based on the gene products of 29 presumptive loci do not support recognition of the lsla Monserrate population as a valid species. It is recommended that the insular population be referred to P. decurtatus arenicolus. INTRODUCTION The leafnose snakes, Phyllorhynchus Stejneger, occur in xeric regions of western North America. Two, or sometimes three, species are recognized. The saddle leafnose snake, P. browni Stejneger, occurs in southcentral Arizona and adjacent northcentral Sonora, Mexico, and in an apparently isolated geographic region in southern Sonora, Mexico. The most widespread species, the spotted leafnose snake, P. decurtatus Cope, is recorded from central Sonora, Mexico along the Gulf of California coast north to southern Nevada as well as throughout xeric and subtropical regions of southern California and Baja California Norte and Sur, Mexico, including three islands in the Gulf of California: lslas San Marcos, San Jose, and Cerralvo. The third form, P. arenicolus Savage and Cliff, occurs on lsla Monserrate in the Gulf of California. Phyllorhynchus arenicolus has been accorded specific status on three occasions since its description by Savage and Cliff (1954); Cliff (1954); Smith and Taylor (1966), Powers and Banta (1974). However, this insular population has also been considered a subspecies of P. decurtatus by Wright and Wright (1957), Soule and Sloan, (1966) and Smith and Smith (1976). Unfortunately, the latter authors did not justify their taxonomic application; the trinomial was simply listed. In the absence of such justification and with present taxonomic discordance, we found it worthwhile to reevaluate the status of Savage and Cliff's insular form. Our recent collecting efforts on lsla Monserrate and lsla San Marcos provided us with a single individual from the former island and four specimens from the latter, where it had not been previously reported. In addition, 5 leafnose snakes (P. decurtatus) were collected on the peninsula of Baja California. Thus we had the opportunity to examine the relative degree of differentiation among the peninsular and two insular populations as a test of their taxonomic status. One of the populations assigned to P. decurtatus is on a land bridge island, lsla San Marcos, that had a terrestrial connection with the peninsula as late as 12,000 years ago (Soule and Sloan, 1966; Wilcox, 1978). The other insular population, P. arenicolus, is on an island of questionable age, lsla Monserrate. The lsla San Marcos population provides a valuable outside reference point as most populations of reptiles on land bridge islands have hardly differentiated from peninsular populations and thus have not been accorded species status (Murphy and Ottley, 1981; Soul6 and Sloan, 1966). Indeed, only two of well over 150 reptile populations studied on land bridge islands in the Gulf of California have been accorded specific status: Chilomeniscus punctissimus and Masti-

264 ROBERT W. MURPHY AND JOHN R. OlTLEY cophis barbouri, both from lslas Espiritu Santo and Partida Sur (Murphy and Ottley, 1981). (The chuckwallas, genus Sauromalus, are not considered in this estimate of island endemicity as we feel their systematics is in need of reexamination.) To examine the relative degrees of differentiation among the populations in question, we undertook a genetic analysis employing electrophoretic methods. To ascertain the significance of any noted electrophoretic differences between the three populations of leafnose snakes we have formed a null hypothesis. In general, conspecific populations of vertebrates have very low levels of electrophoretic differences and henceforth high similarity; two conspecific populations without restrictions to gene flow should be essentially genetic equivalents. Thus our null hypothesis states that our populations are conspecific and accordingly are hardly differentiated electrophoretically. To reject our null hypothesis we must show significant genetic differentiation between populations of Phyllorhynchus on lsla Monserrate and the adjacent peninsula of Baja California Sur. MATERIALS AND METHODS Specimens were collected at night on the islands by lantern walking, and on the peninsula by a combination of lantern walking and road driving. The specimens were sacrificed within three days of capture by injection of sodium pentabarbitol. Heart, kidney, liver and muscle (skeletal) tissues were removed and initially frozen in liquid nitrogen with subsequent laboratory storage of the tissues at -42 C. The voucher specimens were preserved in the field with 10% buffered formalin and deposited in the herpetological collections of Brigham Young University, Provo, Utah (BYU), California Academy of Sciences, San Francisco (CAS) and the Los Angeles County Museum of Natural History (LACM). Four specimens were collected in Arroyo de la Taneria, lsla San Marcos, Baja California Sur, Mexico (BYU 33682-83, 33685-86), one on the northcentral side of lsla Monserrate, Baja California Sur, Mexico (BYU 33715), two from 4.2 km (by road) west of El Arco, Baja California Sur, Mexico (BYU 33729-30), one from 1.6 km (by road) south of El Cien on Mexico Hwy 1, Baja California Sur, Mexico (CAS 147694), and one from 22.9 km (by road) north of La Paz on Mexico Hwy. 1, Baja California Sur, Mexico (LACM 128278). Tissues from all samples were thawed, minced with scissors, and refrozen in distilled, deionized water for 18 hrs. The tissue samples were then thawed and centrifuged at 31,000 g at 2 C for 30 min. The supernatant fractions were saved for electrophoretic analysis. Horizontal starch gel electrophoresis (Selander et al., 1971 and Yang et al., 1974) was performed using multiple buffer systems and various potential voltages (Table 1) to separate a number of enzymatic and nonenzymatic presumptive gene products for study. Electromorph data were treated as the products of codominant alleles at a given locus. Electromorphs sharing common electrophoretic mobility were scored as homologous products of a given allele. Enzyme nomenclature used in this paper follows the recommendations of the Nomenclature Committee of the International Union of Biochemistry (1979). The products of presumptive gene loci controlling expression in the following systems were scored from staining procedures slightly modified from Selander et al. (1971) [synonyms in brackets]: soluble and mitochondria1 aspartate aminotrans- TABLE 1. Gel and electrode bath buffers, potential voltage and time used in electrophoretic analysis. Buffer Gel Electrode Potential Duration System Buffer Buffer Reference voltage1 (in hrs.) A Tris-hydrochloric acid Borate Selander et al. (1971) 14.3 2.5 B Lithium hydroxide Lithium hydroxide Selander et al. (1971) 14.3 5.0 C Poulik Borate Selander et al. (1971) 14.3 4.0 D Phosphate-citrate Phosphate-citrate Selander et al. (1971) 5.7 5.0 E Tris-citrate II Tris-citrate I1 Selander et al. (1971) 7.9 5.0 F ASAC ASAC Avise et al. (1975) 10.7 5.0 'Potential Voltage, volts per cm, is maximum; current was adusted to maintain a maximum of 75 ma on systems B and C

PHYLLORHYNCHUS ARENICOLUS STATUS 265 ferase (S-Aat-A, M-Aat-A [GOT]; E.C. 2.6.1.1); TABLE 2. Summary of electrophoretic data. Protein phenoaminopeptidase (AP [LAP]; E.C. 3.4.1 1,I); types are lettered in order of increasing mobility. alcohol deh~drogenase (Adh-A; E'C' 1; protein Buffer peninsula Sari Marcos Monserrate lactate dehydrogenase (Ldh-A, Ldh-B; E.C. 1.1.1.27); soluble and mitochondria1 malate Gpi A I I I dehydrogenase (S-Mdh, M-Mdh; E.C. 1.1.137); Ck-A A I I I glucosephosphate isomerase (Gpi-1 [PGI]; E.C. A 1 1 1 Pep- I 1 1 1 5.3.1.9); phosphoglucomutase (Pgm-A; E.C. Pep-2 A I 1 I 2.7.5.1); isocitrate dehydrogenase (Icdh-1, Icdh- pep-3 A I I I 2 [IDH]; E.C. 1.1.1.41); phosphogluconate ' Gdh D I I I dehydrogenase (Pgdh-A [6-PGD]; E.C. Fum D 1 I I B 1 1 1 1.1.1.44); glycerol-3-phosphate dehydrogenase B 1 1 1 (G-3-pdh-A, G-3-pdh-B [agpd, EGPDH]; E.C. Est-l a 0.8 0.375 I 1.1.1.8); non-specific esterases (Est-1, Est-2); b B 0.2 0.625 - general proteins (Gp-1); fumerate hydratase Est-2 B I I I (Fum-A; E.C. 4.2.1.2). Superoxide dismutase AP B 1 1 1 Ad h B 1 1 1 (Sod-1, Sod-2 [IPO]; E.C. 1.15.1.l) was scored Me B 1 1 1 from gels stained for PGM. The staining pro- GP-I a B I 0.875 0.5 cedures of Shaw and Prasad (1970) were b B - 0.125 0.5 slightly modified to resolve the products of the Ldh-A c I I I following 3 loci: creatine kinase (Ck-A, Ck-C; FiBA a C 1 1 1 0.7 1 1 E.C. 2.7.3.2); glutamate dehydrogenase (Gdh- b c 0.3 - - A; E.C. 1.4.1.2). Peptidases (Pep-1, Pep-2, sod-i c I I I Pep-3; E.C. 3.4.11 or 13) were scored following Sod-2 C I 1 I 1 Murphy and Papenfuss (1980) and L-iditol i:::'; I I F 1 1 1 dehydrogenase expression (L-ldh [SDH]; E.C. M-Mdh-A I 1 I 1.1.1.14) was resolved from a modified stain of s-m~~-a a F 0.7 0.25 1 Lin et al. (1969). The electrophoretic buffer b F 0.3 0.75 - systems (Table 1) used to separate the various Pgdh a E 0.7 1 1 b E 0.3 gene products are given in Table 2. - - L-ldh E 1 1 1 The electromorphic data were encoded as G-3-pdh-A 1 1 1 allelic freqencies to measure Genetic Identity (I) ~-3-pdh-~ E 1 1 1 and Genetic Distance (D) following Nei (1972). RESULTS TABLE 3. Matrix of genetic identity (I) and genetic distance (D) values. I values are to the right of the diagonal, D's are left; values of D are given 2 one standard error. The electrophoretic comparisons made between lslas Monserrate and San Marcos and PopU1ation P s M the peninsula were based on gene products of Peninsula (p) 0.98 0.98 29 scored loci for each individual of each popu- Sari Marcos (S) 0,02 0.03 0.96 lation; the allele frequencies are given in Table lsla Monserrate (M) 0.02 2 0.03 0.04 2 0.04 2. A matrix of I and D values between populations is given in Table 3. Estimates of genic variability for the lsla San Marcos and Peninsular populations based on the observed number of heterozygotes are presented in Table 4. Of the 29 loci scored, none were found to be fixed for alternate alleles among the three populations, and all loci except the following were determined to be monomorphic: Est-1, Pgm-A, S-Mdh-A, Pgdh and Gp-1. Heterozygotes (and number) were detected on the peninsula at Est-1 (I), Pgm-A (I), S-Mdh-A and Pgdh (I), on lsla San Marcos at Est-1 (1) and Gp-1 (I), and on lsla Monserrate at Gp-1 (1); at least one heterozygote was detected at each of the 5 polymorphic loci.

266 ROBERT W. MURPHY AND JOHN R. OTTLEY TABLE 4. Summary of genetic variability in two populations of leafnose snakes. DISCUSSION The absence of any population-specific Peninsula of lsla Baja Sari Marcos alleles suggests that the lsla Monserrate population is genetically quite similar and closely Sample size 5 4 related tothe peninsular population. To assess Frequency of minor 0.1 O.1Z5 the sianificance " of the absence of uniaue. fixed,. allele detection alleles, two comparisons were made: 1) the % loci polymorphic1 13.7 10.3 alleles per IOCUS 1.14,,,, degree of genetic similarity (I) between the land % heterozygosity 4.1 1.7 bridge island, lsla San Marcos, population and per individual2 the ~o~ulation refered to P. arenicolus on lsla ~onkekrate; 2) the genetic differentiation (D) of IA locus was considered polymorphic if more than one allele was detected. P. arenicolus to the peninsular population (P. %ased on the observed number of heterozygotes. decurtatus) in relation to differentiation observed in other reptile groups. 1. Genetic Similariry--As noted, all three populations of leafnose snakes are genetically very similar to one another. From Table 3 it can be seen that the populations of lsla Monserrate and the peninsula and that of lsla San Marcos and the peninsula are essentially identical (I = 0.98); the least amount of similarity is found between the two island populations. However, this variation is insignificant considering the relatively high standard errors of D (Table 3). Moreover, these values are concordant with other values calculated for land bridge island-peninsular reptilian species pairs (Murphy, unpublished data). The result of this genetic identity analysis could be interpreted to support the consideration of P. arenicolus and P. decurtatus as conspecific. But how do these data compare with those for other reptiles and vertebrates? 2. Genetic Differentiation-There have been several reviews of the relative degree of genetic distance (D) between species of vertebrates. In general, a D of 0.2 or greater is measured among sister species of reptiles and other vertebrates, D measures 0.1-0.2 among most subspecies, and D is between 0 and 0.1 for most local populations (Adest, 1977; Avise and Ayala, 1976; and Murphy, unpublished data). From Table 2 it can be seen that all of the Phyllorhynchus populations have a D of 0.02 to 0.04. These values are well within the range of values detected for local populations, not subspecies or species. As with the genetic identity values the examination of the D values strongly suggest that the lsla Monserrate population of leafnose snakes should not be considered a species, nor even a subspecies. One variable which could significantly effect the calculated values of both I and D is sample size. Avise (1974), Gorman and Renzi (1979), Nei (1979) and Nei and Roychoudhury (1974) have shown that an electrophoretic analysis based upon a small number of individuals, indeed a sample size of 1, can yield reasonable estimates of genetic similarity and distance providing the number of loci sampled is large. Indeed, Gorman and Renzi (1979) found an average deviate of D of only 0.03 from the full sample estimates (25 individuals), and 94% of the estimates were within a D value of 0.10. Moreover, they noted in their analysis, based on 22 presumptive gene loci, that most deviates (82%) were overestimates of D (and thus underestimates of I) and that the average deviate increased as the number of loci utilized decreased. We have examined almost 30% more loci than Gorman and Renzi (1979). Consequently, we should have less of a deviate from our value of D between lsla Monserrate and the other populations, and there is at least an 8O0I0 chance that we are overestimating the D between lsla Monserrate leafnose snakes and the population on the peninsula of Baja California. The problem of sample size, however, is not critical to our findings. Of greater importance and significance to our analysis would be the presence of unique, fixed alleles. If an apparent single, unique, fixed allele had been detected in the population of leafnose snakes on lsla Monserrate at any of the 29 loci surveyed, then it would have been necessary to have increased the sample size of at least the peninsular population to identify the frequency of the allele on the peninsula in the event that the unique allele could be found. However, this problem was not encountered; we were

PHYLLORHYNCHUS ARENICOLUS STATUS 267 able to associate every electromorph scored from the island population with one of identical mobility on the peninsula. Thus, since no unique alleles were detected on lsla Monserrate, the sample sizes in this investigation were sufficient. Although there is seemingly very close genetic affinity between the three leafnose snake populations, we do not advocate that a given I or D value be used as an absolute value for making decisions about species or non-species status of allopatric populations. Our null hypothesis is that the lsla Monserrate and peninsular populations of Phyllorhynchus are conspecific. We are unable to reject our null hypothesis on electrophoretic grounds. At the same time, our null hypothesis is not proven. Thus, our electrophoretic data including our I and D estimates cannot be used as "proof" of conspecific status of the two populations. Perhaps insights into potential genetic distinctiveness can be gained from morphological data. It appears as though the lsla Monserrate population is indeed morphologically distinctive although not detectably differentiated genetically. The Phyllorhynchus population on lsla Monserrate is unique from P. d. decurtatus on the peninsula in having a higher number of sex-specific caudal scales (male decurtatus with 33-36 caudals, male arenicolus with 39; female decurtatus with 22-27, female arenicolus with 31; (Murphy and Ottley, personal observation; Savage and Cliff, 1954). Moreover, there are color pattern differences; the body blotches are about twice as wide as interspaces and at least one fourth of the blotches are partially split by a light medial area. These morphological differences may be produced by genetic peculiarities resulting from selection or random drift, but also could be produced by strictly environmental factors such as temperature of egg incubation (Fox, 1948). And indeed, the color pattern difference does not appear to be quite so significant since 5 of 6 leafnose snakes from lsla San Marcos have blotches partially split by a light medial area, although in an average frequency of less than 25%. These morphological differences seem minor and do not seem significant enough to warrant specific recognition alone. Considering these genetic data, recognition of the Monserrate Island population of leafnose snakes as a species seems unwarranted, but subspecific status may be justified on morphological and geographical grounds. The island population is isolated and is morphologically distinct. Accordingly, we recommend that the island population be referred to as Phyllorhynchus decurtatus arenicolus Savage and Cliff. ACKNOWLEDGMENTS The leafnose snakes utilized in this study were imported on Mexico Scientific Collectors Permit No. 65-78-866 issued to RWM, and Permit No. 1-78-832 to JRO by the Direccion General de la Fauna Silvestre. The assistance of Gloria Arzola to one of us (RWM) in obtaining a permit is gratefully acknowledged. For transportation to and from lsla Monserrate we thank Terry A. Vaughan. Mike Mahlstedt and John Cram provided untiring assistance in the field. Field work was supported in part by the Theodore Roosevelt Memorial Fund, University of California Regents Research and Travel Grant No. 780000-07427-5, and University of California Chancellors Patent Fund Grant No. 780000-08613-5 to RWM and by the Associated Students of Brigham Young University to JRO. The genetic analysis was supported by NSF Grant DEB 77-03259 to George C. Gorman. Thanks go to Donald G. Buth, G. C. Gorman, and one anonymous reviewer for critical evaluation of the manuscript. LITERATURE CITED Adest, G. A. 1977. Genetic relationships of the genus Uma (Iguanidae). Copeia 1977:47-52. Avise, J. C. 1974. Systematic value of electrophoretic data. Syst. Zool. 23:465481. and F. J. Ayala. 1976. Genetic differentiation in speciose versus depauperate phylads: evidence from the California minnows. Evolution 30:4&58.

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